T cell play a central role in the human immune system and their depletion leads to increased susceptibility to infections and some malignancies. T cell development requires a period of education in the thymus, an organ that involutes in late adolescence. If T cells suitable for use in human patients could be generated in vitro, it could lead to novel immunologic therapies for infections, immunodeficiencies and malignancies. Existing In vitro systems that support T cell development use co-culture with allogeneic human or animal tissues, making them less suitable for the production of T cells for use in humans. There are remarkable similarities between the epithelial and stromal cells of the thymus and keratinocytes and fibroblasts of skin. I present evidence that human skin cells arrayed on a three-dimensional matrix support the full process of human T cell development. Newly generated T cells are diverse, functionally mature, and tolerant to self-MHC. To extend this work, I propose to 1) determine if monolayer cultures of skin cells can support T cell development, 2) to examine what stages of development are supported by keratinocytes vs. fibroblasts and to determine if skin cells transduced with the Notch ligand delta-like 1 support augmented T cell production, 3) to examine self-tolerance in T cells generated in cultures with skin cells transduced to express AIRE, 4) to demonstrate that fully autologous T cells can be generated using skin and bone marrow from a single individual and 5) to examine the ability of T cells generated in skin cell cultures to reconstitute immunodeficient mice. My goal is to develop a system in which samples of adult skin and bone marrow can be used to generate autologous, self-tolerant T cells for the treatment of human disease. This proposal describes a mentored four-year career development plan designed to facilitate my transition from post-doctoral fellow'to independent investigator. It takes advantage of the many resources available to me in the laboratory of Dr. Thomas Kupper and in the Harvard Medical School environment.